Monocytic myeloid‐derived suppressor cells regulate T‐cell...

Basic

1022 Carl Fortin et al. Eur. J. Immunol. 2017. 47: 1022–1031DOI: 10.1002/eji.201646797

Immunity to infection

Research Article

Monocytic myeloid-derived suppressor cells regulateT-cell responses against vaccinia virus

Carl Fortin1, Yiping Yang1,2 and Xiaopei Huang1

1 Division of Hematologic Malignancies and Cellular Therapy, Department of Medicine, DukeUniversity Medical Center, Durham, NC, USA

2 Division of Hematologic Malignancies and Cellular Therapy, Department of Immunology,Duke University Medical Center, Durham, NC, USA

Vaccinia virus (VV) can potently activate NK- and T-cell responses, leading to efficient viralcontrol and generation of long-lasting protective immunity. However, immune responsesagainst viral infections are often tightly controlled to avoid collateral damage and sys-temic inflammation. We have previously shown that granulocytic myeloid-derived sup-pressor cells (g-MDSCs) can suppress the NK-cell response to VV infection. It remainsunknown what regulates T-cell responses to VV infection in vivo. In this study, wefirst showed that monocytic MDSCs (m-MDSCs), but not g-MDSCs, from VV-infectedmice could directly suppress CD4+ and CD8+ T-cell activation in vitro. We then demon-strated that defective recruitment of m-MDSCs to the site of VV infection in CCR2−/− miceenhanced VV-specific CD8+ T-cell response and that adoptive transfer of m-MDSCs intoVV-infected mice suppressed VV-specific CD8+ T-cell activation, leading to a delay inviral clearance. Mechanistically, we further showed that T-cell suppression by m-MDSCsis mediated by indication of iNOS and production of NO upon VV infection, and that IFN-γis required for activation of m-MDSCs. Collectively, our results highlight a critical role form-MDSCs in regulating T-cell responses against VV infection and may suggest potentialstrategies using m-MDSCs to modulate T-cell responses during viral infections.

Keywords: Immune regulation � Monocytes � T cells

� Additional supporting information may be found in the online version of this article at thepublisher’s web-site

Introduction

Vaccinia virus (VV), the most studied member of the poxvirus fam-ily, is the live vaccine responsible for the successful elimination ofsmallpox worldwide [1]. This success has led to the developmentof recombinant VV as a vaccine vehicle for infectious diseases andcancer [2, 3]. This unique potency of VV is, in large part, due toits ability to elicit strong and long-lasting protective T-cell immu-

Correspondence: Dr. Yiping Yange-mail: [email protected]

nity [4, 5]. Recent studies have also shown that VV can efficientlyactivate the innate immune system through both TLR-dependentand -independent pathways [6, 7], both of which are critical forCD8+ T-cell responses to VV infection in vivo [8, 9]. Furthermore,VV can efficiently activate NK cells and the activated NK cellsmigrate to the site of infection, contributing to the initial viralcontrol [10–14].

Myeloid-derived suppressor cells (MDSCs), a heterogeneouspopulation of immature myeloid cells, was first shown to playan important role in the regulation of immune responses incancer patients in that the accumulation of MDSCs at tumorsites suppresses antitumor immunity and promotes tumor growth

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Eur. J. Immunol. 2017. 47: 1022–1031 Immunity to infection 1023

Figure 1. m-MDSCs suppress T-cell pro-liferation in vitro. (A and B) m-MDSCspurified from PEs of VV-infected micewere cultured with purified naıve CD4+

(CD4) or CD8+ (CD8) T cells labeled withCFSE at different m-MDSCs to T-cell ratios(m-MDSC:T cells), in the presence of 1μg/mL of anti-CD3 and anti-CD28 anti-bodies. Some T cells were incubated withmedium only (Control). Some T cells wereincubated with g-MDSCs at g-MDSC:T-cell ratio of 2:1. T-cell proliferation wasdetermined 72 h later. (A) Flow cytometryplots showing the percentage of CFSElow

cells representing proliferating T cells. (B)The mean percentages ± SD of CFSElow

cells among total CD4+ or CD8+ T cellsare shown (n = 4). (C and D) m-MDSCswere cultured with CFSE-labeled naıveHA-specific CD4+ (6.5) or CD8+ (Clone 4)T cells at different m-MDSCs to T-cellratios (m-MDSC:T cells), in the presenceof class I or class II HA peptide-pulsedCD11c+ DCs. Some 6.5 and Cone 4 T cellswere incubated with unpulsed CD11c+

DCs (Unpulsed). Some T cells were incu-bated with g-MDSCs at g-MDSC:T-cellratio of 2:1. Proliferation was assessed72 h later. (C) Flow cytometry plots show-ing the percentage of CFSElow cells. (D)The mean percentages ± SD of CFSElow

cells among total 6.5 or Clone 4 T cells areshown (n = 3). Asterisks indicate the p-value (unpaired Student t-test) comparedto the 0:1 ratio: *p < 0.05; **p < 0.01;***p < 0.001. Data shown are from singleexperiments representative of three inde-pendent experiments.

[15, 16]. Since then, extensive studies have established a criti-cal role for MDSCs in the regulation of T-cell responses withinthe tumor microenvironment [17, 18]. There are two subsetsof MDSCs in mice: granulocytic MDSCs (g-MDSCs) are definedby CD11b+Ly6CloLy6G+; whereas monocytic MDSCs (m-MDSCs)have a phenotype of CD11b+Ly6ChiLy6G− [18]. It has recentlybecome clear that these two populations have distinct cellular tar-gets and suppressive capacities [19].

The expansion of MDSCs has also been observed in responseto viral infections [20–24]. In a murine model of VV infection, wehave recently shown that both g-MDSCs and m-MDSCs accumu-lated at site of infection and g-MDSCs are critical for the regulationof the NK-cell response to VV infection through the production ofreactive oxygen species [23]. However, it remains unknown withregard to the role of m-MDSCs in immune responses against VVinfection in vivo.

In this study, we evaluated whether m-MDSCs could influenceT-cell responses to VV infection in vivo. We first showed that m-MDSCs, but not g-MDSCs, from VV-infected mice could directlysuppress the activation of CD4+ and CD8+ T cells in vitro. We thenfound that recruitment of m-MDSCs to the site of VV infection isdependent on CCR2 and that defective m-MDSC recruitment in

CCR2−/− mice led to enhanced VV-specific CD8+ T-cell response.Furthermore, adoptive transfer of m-MDSCs into VV-infected micesignificantly suppressed the VV-specific CD8+ T cells and delayedviral clearance, suggesting an important role for m-MDSCs in reg-ulating T-cell responses against VV infection. We further demon-strated that induction of inducible nitric oxide synthase and theproduction of nitric oxide by m-MDSCs were required for the sup-pression of T-cell responses. Finally, we showed that the suppres-sive capacity of m-MDSC is dependent on IFN-γ.

Results

m-MDSCs inhibit T-cell proliferation in vitro

We have shown previously that g-MDSCs, but not m-MDSCs, ham-pered the NK-cell response to VV infection [23]. However, sinceboth m-MDSCs and g-MDSCs accumulated in the peritoneal cavityin response to VV infection intraperitoneally, we hypothesized thatm-MDSCs could regulate T-cell responses at the site of VV infec-tion. To address this, we utilized a previously described in vitroT-cell coculture system [9]. We found that addition of m-MDSCs


1024 Carl Fortin et al. Eur. J. Immunol. 2017. 47: 1022–1031

Figure 2. Impaired migration of m-MDSCs to the site of VV infectionin CCR2−/− mice. WT and CCR2−/− mice were infected with 2 × 106 PFUof VV i.p., or left uninfected (Naıve). Seven days later, cells from spleen(A and B) and peritoneal cavity exudates (PE; C and D) were stainedwith anti-CD11b, anti-Ly6G, and anti-Ly6C. (A and C) Flow cytome-try plots showing the percentage of m-MDSCs (Ly6ChiLy6G−) and g-MDSCs (Ly6CloLy6G+) among CD11b+ cells. (B and D) The mean cellnumber ± SD of m-MDSCs or g-MDSCs among total cells is shown(n = 5). Asterisks indicate the p-value (unpaired Student t-test) com-pared to the VV-infected WT group: **p < 0.01; ***p < 0.001. Data shownare from single experiments representative of three independentexperiments.

from VV-infected mice to T-cell cultures markedly suppressedthe proliferation of both CD4+ and CD8+ T cells in response tostimulation with anti-CD3 and anti-CD28 antibodies in a cell dose-dependent manner (Fig. 1A and B). In contrast, no T-cell suppres-sion was observed when g-MDSCs (with g-MDSC to T-cell ratio

of 2:1) were added to the culture (Fig. 1B). To address whetherm-MDSCs were able to suppress antigen-specific T-cell responses,we used influenza hemagglutinin-specific CD4+ and CD8+ T cellsderived from 6.5 and Clone 4 HA-TCR transgenic mice, respec-tively. Similarly, addition of m-MDSCs, not g-MDSCs, significantly(p < 0.01) inhibited the proliferation of HA-specific CD4+ andCD8+ T cells in response to stimulation with their respective cog-nate peptides (Fig. 1C and D). These results indicate that m-MDSCs could directly suppress antigen-specific and -nonspecificT-cell responses in vitro.

Defective m-MDSC recruitment to infection site inCCR2−/− mice enhances VV-specific T-cell response

It has been shown that m-MDSCs express CCR2 [25, 26] andthat CCR2−/− MDSCs had an impaired migration to the tumormicroenvironment [26]. We thus used CCR2−/− mice to evaluatethe role of m-MDSCs in CD8+ T-cell response to VV infection invivo. We first determined whether there was a defect in m-MDSCrecruitment in CCR2−/− mice in response to VV infection. Wefound that the percentage (Fig. 2A and C) and absolute number(Fig. 2B and D) of m-MDSCs in the spleen (Fig. 2A and B) andperitoneal exudates (PEs, Fig. 2C and D) were markedly lowerin VV-infected CCR2−/− mice than the infected wild-type con-trols, suggesting that the migration of m-MDSCs to the site of VVinfection is severely compromised. However, the recruitment ofg-MDSCs was not affected (Fig. 2).

We next investigated whether defective recruitment of m-MDSCs to the site of infection in CCR2−/− mice affected the CD8+

T-cell response to VV infection. Using an immunodominant VV-derived epitope peptide, B8R20-27 [27], we assessed the productionof IFN-γ by VV-specific CD8+ T cells. We found that there was aslight increase in the percentage of IFN-γ+CD8+ T cells in spleensof infected CCR2−/− mice compared to WT mice (Fig. 3A and B).However, the frequency of IFN-γ+CD8+ T cells in peritoneal cavitywas significantly (p < 0.01) increased in infected CCR2−/− mice(Fig. 3B). Collectively, these results indicated that defective migra-tion of m-MDSCs to the site of VV infection enhanced VV-specificCD8 T-cell response, suggesting an inhibitory role for m-MDSCsin VV-specific CD8+ T-cell response in vivo.

Figure 3. Enhanced CD8+ T-cell response to VVinfection in CCR2−/− mice. WT and CCR2−/− micewere infected with 2 × 106 PFU of VV i.p., or leftuninfected (Naıve). Seven days later, cells fromspleen and peritoneal cavity exudates (PE) werestimulated with 2 μg/mL B8R20-27 peptide andstained for IFN-γ production by CD8+ T cells intra-cellularly. (A) Flow cytometry plots showing thepercentage of IFN-γ+ B8R-Specific CD8+ T cellsamong total cells. (B) The mean percentage ± SD ofIFN-γ+ B8R-Specific CD8+ T cells among total cellsis provided (n = 6). Asterisks indicate the p-value(unpaired Student t-test) compared to the infected(VV) WT group: **p < 0.01. Data shown are fromsingle experiments representative of three inde-pendent experiments.



Figure 4. m-MDSCs suppress VV-specific CD8+ T-cell response anddelay viral clearance in vivo. 104 purified naıve HA-specific CD8+ Tcells (Thy1.1+) were adoptively transferred into B10.D2 mice (Thy1.2+),which were subsequently infected with 5 × 106 PFU of a recombinantVV-HA. Five days later, these mice received m-MDSCs or g-MDSCs ata dose of 0.5 × 106 or 1 × 106 cells purified from another cohort ofVV-infected mice or a vehicle control. Two days later after transfer,splenocytes were analyzed for (A and B) the presence of total andIFN-γ+ HA-specific CD8+ T cells by flow cytometry, as well as for (C)the detection of VV DNA by quantitative real-time PCR. (A and B) Themean cell numbers ± SD of (A) total and (B) IFN-γ+ Thy1.1+CD8+ T cellsamong total cells are shown (n = 4). (C) Relative viral DNA amount ±SD (n = 4). Samples were normalized to β-actin gene. Asterisks indicatethe p-value (unpaired Student t-test) compared to the control group:***p < 0.001. Data shown are from single experiments representative ofthree independent experiments.

m-MDSCs suppress the CD8+ T-cell response to VVinfection in vivo

To further support the inhibitory role of m-MDSCs, we askedif adoptive transfer of m-MDSCs suppressed the CD8+ T-cellresponse to VV infection in vivo. We used a previously describedmodel in which CD8+ T cells from Clone 4 HA-TCR transgenicmice were adoptively transferred into congenic mice, followed byinfection with VV-HA [28, 29]. Five days after infection, mice were

adoptively transferred with m-MDSCs purified from VV-infectedmice. We found that a significant (p < 0.001) reduction in thenumber of total (Fig. 4A) and IFN-γ+ (Fig. 4B) clonotypic HA-specific CD8+ T cells at a dose-dependent fashion, compared tovehicle transferred control mice. By contrast, no reduction in HA-specific CD8+ T cells was observed in mice received g-MDSCs (Fig.4A and B). The suppression of virus-specific T-cell response by m-MDSCs was associated with an increase in VV DNA (Fig. 4C).These results suggest that m-MDSCs can directly suppress VV-specific CD8+ T-cell response, leading to a delay in viral clearancein vivo.

m-MDSC iNOS induction and NO production in VVinfection are critical for their suppressive activity

How do m-MDSCs from VV-infected mice suppress T-cellresponses? The induction of iNOS and the production of NO, theinduction of arginase-1 activity, and the production of ROS havebeen implicated in the suppressive activity mediated by MDSCs onanti-tumor T-cell responses [30]. To examine the potential roles ofiNOS, arginase-1, and ROS, we added their respective inhibitorsto the m-MDSC-T cell coculture system. These inhibitors includeL-NMMA for iNOS, nor-NOHA for arginase-1, SOD for ROS. Wefound that L-NMMA could completely reverse m-MDSC-mediatedsuppression on the proliferation of CD4+ and CD8+ T cells, andnor-NOHA could partially reverse the suppression, whereas SODhad no effects on m-MDSC-mediated suppression (Fig. 5). Thereversal of suppression on T cells by L-NMMA was associated witha marked reduction of NO in the culture (data not shown), sug-gesting a role for NO in mediating the suppression. These resultsindicated that m-MDSC-mediated suppression on T-cell activity ismainly mediated by the induction of iNOS.

We next examined whether VV infection induced m-MDSCs toupregulate iNOS and produce NO. To address this question, micewere injected with VV or LPS, i.p. Three days later, m-MDSCsor g-MDSCs were purified from peritoneal cavity exudates andassayed for iNOS induction and NO production. We found thatm-MDSCs from VV-infected, but not LPS-treated, mice had a sig-nificant (p < 0.001) increase in the expression iNOS (Fig. 6A)and production of NO (Fig. 6B), compared to naıve control. How-ever, no significant induction of iNOS or NO by g-MDSCs wasobserved (Fig. 6). These results suggest that VV infection prefer-entially induced m-MDSCs to upregulate iNOS and produce NO.

IFN-γ activates m-MDSC activity

We next investigated what regulates the activity of m-MDSCsin response to VV infection. IFN-γ has been implicated in theactivation of m-MDSC within the tumor microenvironment [25].Since IFN-γ is produced in high quantity during viral infection,we hypothesized that IFN-γ may be required for the activation ofm-MDSC elicited upon VV infection. To test this, we coculturedm-MDSC from peritoneal cavity with CD4+ or CD8+ T cells in the



Figure 5. Suppression by m-MDSCs is mediated by iNOS. (A and B) m-MDSCs purified from VV-infected mice were cocultured with naıve CFSE-labeled CD4+ (CD4) or CD8+ (CD8) T cells at a 2:1 m-MDSC to T-cell ratio (+m-MDSC), in the presence of 1 μg/mL of anti-CD3 and anti-CD28antibodies (CD3/CD28). Some T cells were incubated with media only (Control). Where indicated, SOD, L-NMMA, or nor-NOHA was added tothe culture. Proliferation was determined 72 h later. (A) Flow cytometry plots showing the percentage of CFSElow cells representing proliferatingT cells. (B) The mean percentages ± SD of CFSElow cells among total CD4 or CD8 cells are shown (n = 4). (C and D) m-MDSCs were coculturedwith CFSE-labeled naıve HA-specific CD4+ (6.5) or CD8+ (Clone 4) T cells at a 1:2 m-MDSC to T-cell ratio (m-MDSC:T cells) in the presence ofHA-pulsed CD11c+ DCs (Pulsed). Some 6.5 and Clone 4 cells were incubated with control CD11c+ DCs (Unpulsed). Where indicated, SOD, L-NMMA,or nor-NOHA was added to the culture. Proliferation was assessed 72 h later. (C) Flow cytometry plots showing the percentage of CFSElow cells.(D) The mean percentages ± SD of CFSElow cells among total 6.5 or Clone 4 T cells are shown (n = 4). Asterisks indicate the p-value (unpairedStudent t-test) compared to the CD3/CD28 (B) or the pulsed (D) group: *p < 0.05; **p < 0.01; ***p < 0.001. Data shown are from single experimentsrepresentative of three independent experiments.

presence of a blocking anti-IFN-γ mAb. We showed that the addi-tion of anti-IFN-γ mAb could reverse the suppression of CD4+ andCD8+ T-cell proliferation by m-MDSCs (Fig. 7A and B). To addressthe role of IFN-γ in m-MDSC activity in response to VV infection invivo, m-MDSC purified from VV-infected WT and IFN-γ−/− micewere measured for NO production. We found that NO productionby m-MDSC from IFN-γ−/− mice was significantly (p < 0.001)reduced (Fig. 7C), compared to that from WT mice.

To further test whether IFN-γ is sufficient to induce NO produc-tion, m-MDSCs or g-MDSCs harvested bone marrow of naıve micewere cultured in the presence of IFN-γ for 24 h and assayed for theproduction of NO. Indeed, we found that addition of IFN-γ signif-icantly (p < 0.001) induced the production of NO by m-MDSCs,but not g-MDSCs (Fig. 7D). Taken together, these results supportthat IFN-γ is required for the suppressive capacities of m-MDSCupon VV infection.

Discussion

In this study, we showed that m-MDSCs from VV-infected micecould directly suppress the activation of CD4+ and CD8+ T cellsin vitro. We also found that defective recruitment of m-MDSCs

to the site of VV infection in CCR2−/− mice enhanced VV-specificCD8 T-cell response. Furthermore, adoptive transfer of m-MDSCsinto VV-infected mice significantly suppressed the activation ofVV-specific CD8 T cells and delayed viral clearance, suggestingan important role for m-MDSCs in suppressing T-cell responsesagainst VV infection. In addition, we demonstrated that m-MDSC-mediated suppression on T cells was mediated by iNOS andNO, and that IFN-γ is required for the suppressive capacity ofm-MDSCs.

Studies have shown that VV can efficiently activate the innateimmune system through both TLR-dependent and -independentpathways [6, 7], leading to potent activation of T-cell responses toVV infection in vivo [8, 9]. VV infection can also efficiently activateNK cells [10–14]. Thus, VV infection is capable of activating boththe innate and the adaptive immune responses for effective viralcontrol. However, immune responses against viral infections areoften tightly regulated to avoid collateral damage and systemicinflammation. We have recently shown that g-MDSCs and ROSare critical for the control of NK-cell activation in response to VVinfection [23]. We now show that T-cell responses to VV infectionare negatively regulated by m-MDSCs. This is mediated by theinduction of iNOS and production of NO by m-MDSCs upon VVinfection.



Figure 6. Induction of iNOS and production of NO by m-MDSCs uponVV infection. Mice were injected with 2 × 106 PFU of VV, or 10 μg ofLPS, i.p., or left untreated (Naıve). Three days later, m-MDSCs or g-MDSCs were purified from peritoneal cavity exudates and assayed foriNOS induction and NO production. (A) Total RNA was isolated andthe expression of iNOS was determined by real-time quantitative PCR.Relative iNOS mRNA abundance ± SD is shown (n = 3). Samples werenormalized to β-actin mRNA. (B) Cells were incubated in medium foradditional 24 h before determining NO production with Griess’ reagent.The mean NO production ± SD is shown (n = 3). The asterisk indicatesthe p-value (unpaired Student t-test) compared to naıve m-MDSCs:***p < 0.01. Data shown are from single experiments representative oftwo independent experiments.

ROS, iNOS/NO, and arginase-1 have been shown to medi-ate suppression on anti-tumor T-cell responses by MDSCs [30].In a model of viral infection, here we show that it is m-MDSCsmediated by iNOS/NO that suppress T-cell responses, whereas g-MDSCs mediated by ROS regulate the NK-cell response [23]. Inter-estingly, g-MDSCs even at a high dose could not suppress T-cellresponses both in vitro and in vivo. The reason for the differentialregulation of NK versus T-cell responses during VV infection bydifferent MDSC subsets in vivo is not entirely unclear, but couldbe related to the timing for induction of suppressive capacitiesof MDSC subsets; the kinetics of NK- and T-cell activation; andthe involvement of different signaling pathways for the respectivesuppression. It has been shown that NO can both directly and indi-rectly target T cells to modulate their activation. NO can disruptthe binding between TCR and MHC by the nitration of tyrosineresidues present in the TCR–CD8 complex, leading to a reductionin T-cell responsiveness [31, 32]. Alternatively, NO can suppressIL-2R signaling, resulting in inhibition of T-cell activation and pro-liferation [33]. Future studies are needed to further investigatethe mechanisms underlying the suppression by different subsetsof MDSCs.

Our observation that migration of m-MDSCs to the site of VVinfection is dependent on CCR2 is consistent with a previous reportthat CCR2 is required for the recruitment of immunosuppressivemonocytes in response to MCMV infection [34]. However, studieshave shown that migration of monocytes to the respiratory tractupon infection with modified vaccinia Ankara is related to CCR1and CCL2 [35, 36]. The reason for the differences remains unclear,but could be related to the complexity of poxviruses that encodea series of genes responsible for various mechanisms of immunemodulation.

Our results that T-cell responses to VV infection is regu-lated by m-MDSCs are consistent with previous observations on

Figure 7. IFN-γ is required for the activation of m-MDSCs. (A and B)Purified naıve CD4+ (A) or CD8+ (B) T cells were labeled with CFSE andcocultured at a 2:1 m-MDSC to T-cell ratio (+m-MDSC) with purified m-MDSC, in the presence of 1 μg/mL of anti-CD3 and anti-CD28 antibodies(CD3/CD28). Some CD4+ and CD8+ cells were incubated with mediaonly (Control). Where indicated, 10 μg/mL of a blocking anti-IFN-γ (α-IFNγ), or an isotype control (Isotype), was added to the culture. T-cellproliferation was determined 72 h later. The mean percentages ± SDof CFSElow cells among total CD4+ or CD8+ T cells are shown (n = 3).Asterisks indicate the p-value (unpaired Student t-test) compared tothe isotype control group to show rescue of proliferation: *p < 0.05;**p < 0.01. (C) WT and IFN-γ−/− mice were infected with 2 × 106 PFU ofVV i.p. Three days later, m-MDSCs were purified from peritoneal cavityexudates and incubated in medium for 24 h before determining NOproduction with Griess’ reagent. The mean NO production ± SD by m-MDSCs is shown (n = 3). The asterisk indicates the p-value compared toWT group: ***p < 0.01. (D) m-MDSCs or g-MDSCs harvested BM of naıvemice were cultured in the presence of IFN-γ (40 U/mL) for 24 h andassayed for the production of NO. The mean NO production ± SD isshown (n = 3). The asterisk indicates the p-value (unpaired Student t-test) compared to the medium control: ***p < 0.01. Data shown are fromsingle experiments representative of three independent experiments.

the role of m-MDSCs in T-cell responses in models of varioustumors [25, 37, 38], experimental autoimmune encephalomyeli-tis [39], graft versus host diseases [40], as well as other viral infec-tions [20–24]. In addition, a recent report has shown that modifiedvaccinia Ankara-based vaccine can induce myeloid cell expansionthat restricts protective CD8 T-cell immunity to SIV [41]. Theseobservations suggest inflammatory signals that drive the activa-tion of m-MDSCs and production of NO during viral infections areprobably similar to other chronic inflammatory conditions, such astumor microenvironment, autoimmunity, and allogeneic rejection.What could drive the production of NO by m-MDSCs in responseto VV infection? Here, we provided evidence that IFN-γ producedduring VV infection is critical for NO production and the suppres-sive function of m-MDSCs both in vitro and in vivo. It has beenshown that IFN-γ is crucial for the control of viral infection andtumor growth [42–44] and IFN-γ can iNOS in a STAT1-dependent



manner, leading to NO production [45, 46]. Thus, IFN-γ producedby activated T cells during VV infection could contribute to theinduction of iNOS and production of NO by m-MDSCs, leading tothe control of potentially overwhelming T-cell activation in vivo.

In summary, we have provided evidence that m-MDSCs cansuppress T-cell responses to VV infection. Mechanistically, m-MDSC-mediated suppression on T cells is mediated by iNOS andNO, and that IFN-γ is required for the suppressive capacity ofm-MDSCs. Our study may suggest potential strategies using m-MDSCs to modulate T-cell activity for potential therapeutic bene-fits in viral infections.

Materials and methods

Mice

C57BL/6 (B6) mice were purchased from the National CancerInstitute (Frederick, MD, USA). B10.D2 and CCR2−/− B6 micewere purchased from The Jackson Laboratory (Bar Harbor, ME,USA). The clone 4 influenza HA-TCR transgenic mice that expressa TCR recognizing a Kd-restricted HA epitope (518IYSTVASSL526)were provided by Dr. L. Sherman (The Scripps Research Insti-tute, La Jolla, CA, USA) [47]. The 6.5 TCR-HA transgenic micethat express a TCR recognizing an I-Ed–restricted HA epitope(110SFERFEIFPKE120) were provided by Dr. H. von Boehmer (Har-vard University, Boston, MA, USA) [48]. These mice were back-crossed onto the Thy1.1+ B10.D2 background. All mice used inthis study were between 7 and 10 week of age. Experimental pro-cedures were performed in accordance with protocols approvedby the Institutional Animal Care and Use Committee of Duke Uni-versity (Durham, NC, USA).

Antibodies and flow cytometry

Allophycocyanin-conjugated anti-CD11b (clone M1/70), PE-conjugated anti-CD8a (clone 53–6.7), PE-Cy5-conjugated anti-CD4 (clone RM4-5), FITC-conjugated anti-IFN-γ (clone XMG1.2),PE-conjugated anti-CD90.1 (Thy1.1; clone OX-7), PE-conjugatedLy6G (clone 1A8), PE-Cy5-conjugated anti-CD8a (clone 53–6.7),FITC-conjugated Ly6C (clone AL-21), purified NA/LE anti-MouseCD3e (clone 145-2C11), purified anti-mouse CD28 (clone 37.51),purified NA/LE anti-mouse IFN-γ (clone R4-6A2), and purifiedNA/LE rat IgG1κ isotype control (R3-34) were purchased from BDBiosciences (San Diego, CA, USA). FACSCanto (BD Biosciences)was used for flow cytometry event collection, which was analyzedusing FACSDiva software (BD Biosciences).

Vaccinia virus

The Western Reserve strain of VV was purchased from AmericanType Culture Collection (Manassas, VA, USA). VV was grown in

TK-143B cells (American Type Culture Collection) and purified bya 35% sucrose cushion as described [49]. The titer was determinedby plaque assay on TK-143B cells, and VV was stored at −80°Cuntil use. For in vivo studies, 2 × 106 PFU of live VV in 0.05 mL1 mM Tris (pH 9) was injected into mice i.p. The recombinant VVencoding HA (VV-HA) was previously described and 5 × 106 PFUof live VV-HA was used for in vivo studies [50].

In vitro T-cell proliferation and suppression assays

For antigen-specific T-cell proliferation and suppression assays,naıve clone 4 CD8+ and 6.5 CD4+ T cells purified from clone 4 and6.5 HA-TCR transgenic mice, respectively, were first labeled with3.3 mM carboxy-fluorescein diacetate, succinimidyl ester (CFSE;Invitrogen, Grand Island, NY, USA). CFSE-labeled T cells (2 × 105)were then cultured in the presence of HA class I [HA518–526 (IYST-VASSL)] or class II [HA110-120 (SFERFEIFPKE)] peptide-pulsedCD11c+ dendritic cells (4 × 104) generated from the BM cells asdescribed [13], in 300 μL T-cell media (RPMI 1640 supplementedwith 5% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 IU/mLstreptomycin, 10 mM HEPES buffer, 0.1 mM nonessentials amino-acids, 1 mM sodium pyruvate, and 50 μM 2-ME) for 72 h.In some experiments, m-MDSCs (CD11b+Ly6G−Ly6Chi) purifiedfrom PEs of B10.D2 mice infected with 2 × 106 PFU of VV for72 h, were added to the coculture at the indicated m-MDSC:T-cellratio.

For polyclonal proliferation assays, CD8+ T cells and CD4+

T cells were purified from naıve B10.D2 mice and labeled withCFSE as described above. CFSE-labeled T cells were then culturedin the presence of 1 μg/mL soluble anti-CD3 and 1 μg/mL solubleanti-CD28, and M-MDSCs in 300 μL T-cell media for 72 h.

In some experiments, 200 U/mL bovine superoxide dismu-tase (SOD; Sigma–Aldrich, St. Louis, MO, USA), 0.5 mM NG-monomethyl-L-arginine (L-NMMA; Calbiochem, San Diego, CA,USA), or 0.5 mM N(ω)-hydroxy-nor-L-arginine (nor-NOHA; Cal-biochem) were added to the coculture to inhibit ROS, iNOS, andarginase-1 activity as described [25, 51]. Finally, the biologicalactivity of IFN-γ was blocked into the coculture by adding a finalconcentration of 10 μg/mL of purified anti-mouse IFN-γ or RatIgG1κ as isotype control.

Measurement of intracellular IFN-γ production byCD8+ T cells

To measure intracellular levels of IFN-γ, 1 × 106 splenocytes orperitoneal cavity exudates were stimulated with either 2 μg/mLB8R20-27 (TSYKFESV) peptide or the Kd-restricted HA518–526 (IYST-VASSL) peptide for 6 h in the presence of 5 μg/mL brefeldin Aat 37°C. After incubation, cells were washed and stained with PE-Cy5-conjugated anti-CD8 or with PE-Cy5-conjugated anti-CD8 andPE-conjugated anti-CD90.1. Cells were then permeabilized usingthe Cytofix/Cytoperm kit (BD Biosciences), subsequently stainedwith FITC-conjugated anti-IFN-γ and analyzed by flow cytometry.



In vivo suppression assay

Naıve CD8 T cells were purified from clone 4 HA-TCR trans-genic mice using anti-CD8 beads according to the manufac-turer’s instructions (Miltenyi Biotec, Auburn, CA, USA). Cells werethen washed twice in cold HBSS and resuspended at 5 × 104

cells/mL in HBSS. Thereafter, 1 × 104 CD8+ T cells were trans-ferred intravenously into B10.D2 mice, which were subsequentlyinfected i.p. with 5 × 106 PFU of VV-HA. Five days after infec-tion, these infected mice received m-MDSCs or g-MDSCs purifiedfrom another cohort of VV-infected B10.D2 mice. Two days later,splenocytes were analyzed for total and IFN-γ+ clonotypic T cellsby intracellular cytokine staining.

Measurement of VV DNA

Total genomic DNA was isolated from the spleen. Real-time quan-titative PCR was used to measure VV genomic DNA using primerslocated in the VV A33R gene. The sequences of the forward andreverse primers for A33R were 5’-TATTACTGACGCCGC TGTTG-3’ and 5’-GTGTTGATGATTCCGCAGTG-3’, respectively. Amountsof VV DNA were normalized to β-actin gene within each sample.Normalized viral DNA value in each sample was calculated as therelative quantity of VV DNA divided by the relative quantity ofβ-actin gene.

iNOS expression by real-time PCR

Total RNA was isolated from 2 × 105 sorted m-MDSCs andg-MDSCs using RNAeasy Mini Kit (Qiagen, Hilden, Germany)according to the manufacturer. First-strand cDNA was performedusing RT kit (Promega, Madison, WI, USA). Quantitative PCRwas carried out with primers for iNOS and relative amounts ofiNOS mRNA were normalized to β-actin. The sequences of theforward and reverse primers for iNOS were GTTCTCAGCCCAA-CAATACAAGA and GTGGACGGGTC GATGTCAC, respectively.

NO production

Equal volumes of culture supernatants (50 μL) and Greissreagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthyl-ethylenediamine dihydrochloride in double-distilledwater, Sigma–Aldrich) were mixed together. After 10 min incuba-tion at room temperature, the absorbance at 550 nm was measuredusing microplate plate reader (Bioteck Instrument, Winooski, VT,USA). Nitrite (NO) concentrations were determined by compar-ing the absorbance values for the test samples to a standardcurve generated by serial dilution of 0.25 mM sodium nitrite[51].

Statistical analysis

A two-sided, unpaired Student t-test with 95% confidence boundand Welch’s correction was used for all statistical analysis, whichwas performed using GraphPad Prism Version 5.0f for Mac OSX (GraphPad Software, San Diego, CA, USA). Significance wasassumed at p < 0.05.

Acknowledgments: We thank Lynn Martinek and Michael Cookfrom the Cancer Center Facility, Duke University, for their excel-lent technical assistance in cell sorting. This work was supportedby the National Institutes of Health Grants CA136934, CA186973,and CA193167 (to Y.Y.). C.F. is supported by a postdoctoral fel-lowship award from the FRSQ (Fonds de recherche du Quebec-Sante).

Conflict of interest: The authors declare no financial or commer-cial conflict of interest.

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Abbreviations: g-MDSC: granulocytic MDSC · MDSC: myeloid-derived

suppressor cell · m-MDSC: monocytic MDSC · PE: peritoneal exudate ·VV: vaccinia virus

Full correspondence: Dr. Yiping Yang, Department of Medicine, DukeUniversity Medical Center, Box 103005, Durham, NC 27710, USAFax: +919-684-9594e-mail: [email protected]

Additional correspondence: Dr. Xiaopei Huang, Department of Medicine,Duke University Medical Center, Box 103005, Durham, NC 27710, USAe-mail: [email protected]

Received: 3/11/2016Revised: 20/2/2017Accepted: 29/3/2017Accepted article online: 6/4/2017


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